ABSTRACT/SUMMARY ? PROJECT 3 (SHOKAT) Unlike glioblastoma (GBM) which has been a challenge to drug development efforts with its multiple sequence and copy number alterations, the relatively stable genome of human supratentorial ependymoma (ST-EPN) contains the fusion gene C11orf95-RELA (RELAFus) that is sufficient to induce ependymoma in mice. Therefore, human ST-EPN may follow a single-hit oncogenesis model such as BCR-ABL-driven chronic myelogenous leukemia (CML). This offers a therapeutic opportunity to treat ST-EPN if an inhibitor of RELAFus function can be developed. We propose a two-pronged approach toward this goal: Aims 1 & 3 target effector pathways induced by the RELAFus product and Aim 2 screens for molecules which directly bind to C11orf95 portion of the fusion protein. Discovery of direct binding molecules to C11orf95 is a challenge because the protein does not contain known drug binding sites. We propose new strategies for covalent drug discovery, some of which the Shokat laboratory applied successfully to the previously ?undruggable? target K-Ras (G12C), providing a new avenue for drug discovery against proteins of unknown structure. To mitigate the risk associated with this ?direct attack? on the oncogenic fusion protein, we also deploy small molecules which target protein kinases essential for RELAFus mediated oncogenesis, as well as drugs which target downstream effector pathways of RELAFus such as the nuclear proteasome. In this project we will identify, target kinases required for RELAFus oncogenesis, and screen for compounds that bind to C11orf95. In addition, we will test candidate repurposing drugs and novel chemical entities in combination with novel delivery methods. Finally, using the RCAS/tva based modeling system developed by the Holland lab, we will determine test small molecule inhibitors we have developed for efficacy in RCAS-RELAFus-driven EPN in vivo mouse model published recently as a collaboration between the Holland and Gilbertson labs.